Silica-based mesoporous carrier and delivery method of using the same

ABSTRACT

Mesoporous carriers for delivering targets into a cell are provided. The mesoporous carriers comprise hollow silica nanospheres (HSN) or mesoporous silica nanoparticles (MSN) and the targets bound to or encapsulated by the hollow silica nanospheres or the mesoporous silica nanoparticles. The targets includes at least two different targets, and the targets may include peptides, proteins, enzymes and/or enzymatic mimetics.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the priority benefits of U.S. provisional application Ser. No. 62/034,181, filed on Aug. 7, 2014, U.S. provisional application Ser. No. 62/034,192, filed on Aug. 7, 2014, and U.S. provisional application Ser. No. 62/034,282, filed on Aug. 7, 2014. The entirety of each of the above-mentioned patent applications is hereby incorporated by reference herein and made a part of this specification.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention generally relates to a mesoporous carrier, in particular, to silica-based mesoporous carriers and the delivery methods by using the silica-based mesoporous carriers.

2. Description of Related Art

Classified by the pore sizes, porous materials can be classified as microporous materials having pore sizes of less than 2 nm, mesoporous materials having pore sizes of 2-50 nm and macroporous materials having pore sizes of greater than 50 nm. The size of the pores and the large surface area of the pores allow the mesoporous materials to be ideal vehicles for carrying chemicals or drugs.

SUMMARY OF THE INVENTION

According to embodiments of the present invention, mesoporous carriers for delivering targets into a cell are provided. The mesoporous carriers comprise hollow silica nanospheres (HSN) or mesoporous silica nanoparticles (MSN) and the targets bound to or encapsulated by the hollow silica nanospheres or the mesoporous silica nanoparticles. The targets includes first targets and second targets, and the first and second targets are different. The targets may include peptides, proteins, enzymes and/or enzymatic mimetics.

According to embodiments of the present invention, a method of delivering targets into a cell is proposed. At first, mesoporous carriers are prepared and provided. The mesoporous carriers comprises hollow silica nanospheres (HSN) or mesoporous silica nanoparticles (MSN) and the targets bound to or encapsulated by the hollow silica nanospheres or the mesoporous silica nanoparticles. The targets include first targets and second targets, and the first and second targets are different. Then the mesoporous carriers contact with the cell by incubating the cell with the mesoporous carriers. The first targets and the second targets are co-delivered into the cell at the same time, as the mesoporous carriers and the targets carried by the hollow silica nanospheres or the mesoporous silica nanoparticles enter into the cell.

In order to make the aforementioned and other features and advantages of the invention more comprehensible, several embodiments accompanied with figures are described in detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification. The drawings illustrate embodiments of the invention and, together with the description, serve to explain the principles of the invention.

FIGS. 1A-1B show the reaction schemes for the PEI-modification of the enzymes SOD and CAT and the encapsulation of PEI-SOD and PEI-CAT within HSN.

FIG. 2 shows the adsorption and desorption isotherms of HSN according to one embodiment of the present invention.

FIGS. 3A-3D show transmission electron microscopy (TEM) images of various enzyme encapsulated HSN.

FIG. 4A shows the relative enzyme activity for different enzyme-encapsulated HSN.

FIG. 4B shows the fluorescence intensity over the wavelength for different enzyme-encapsulated HSN.

FIG. 5 shows the cell viability results using WST-1 assay when exposed to HSN or PEI-SOD/CAT@HSN.

FIG. 6 shows the quantification of fluorescence intensity of oxidized DHE from HeLa cells were treated with various enzyme encapsulated HSN.

FIG. 7 describes the reaction scheme for the conjugation of NF-κB p65 antibody and Cys-TAT peptide to the surface functionalized MSN.

FIGS. 8A-8D show transmission electron microscopy (TEM) images of various functionalized MSN nanoparticles.

FIG. 9A-9C show the results of in vitro pull-down assay of various functionalized MSN nanoparticles.

FIG. 10A shows the conjugation of FMSN-PEG/PEI nanoparticles.

FIG. 10B shows the TEM images of FMSN-PEG/PEI nanoparticles.

FIGS. 11A-11C show the protection effects of co-delivery of TAT-SOD and TAT-GPx into Hela cells.

DESCRIPTION OF THE EMBODIMENTS

Reference will now be made in detail to the present preferred embodiments of the invention, examples of which are illustrated in the accompanying drawings. Wherever possible, the same reference numbers are used in the drawings and the description to refer to the same or like parts. In the following embodiments, one or more silica-based mesoporous carrier materials are described. In the embodiments, the silica-based mesoporous materials may be classified as hollow silica nanospheres (HSN) and mesoporous silica nanoparticles (MSN) and the targets may be carried on the surface of the silica-based mesoporous materials or be encapsulated within the silica-based mesoporous materials. The cited examples, the ingredients, the reaction conditions or parameters illustrated in the examples are merely for illustration purposes, but it is not intended to limit the material or the preparation method by the exemplary embodiments described herein.

The target suitable for being carried by the silica-based mesoporous materials may include an enzymes containing cysteine (thiol group), lysine (amino group), aspartate or glutamate (carboxyl group), a peptide containing cysteine (thiol group), lysine (amino group), aspartate or glutamate (carboxyl group) or an antibody containing cysteine (thiol group), lysine (amino group), aspartate or glutamate (carboxyl group). Alternatively, the target suitable for being carried by the silica-based mesoporous materials may include an enzymes containing polyhistidine-tag (His-tag), a peptide containing polyhistidine-tag or an antibody containing polyhistidine-tag. Herein, the polyhistidine-tag consists of at least six histidine (His) residues.

In one embodiment, hollow silica nanospheres (HSN) with porous silica shells and large interior spaces (cavities) have been synthesized, which is suitable for loading enzymes within the cavities of HSN for intracellular catalysis. A microemulsion method has been developed for synthesizing hollow silica nanospheres (HSN), of which one or more enzymes may be loaded within the cavities. The enzymes may be antioxidant enzymes, including horseradish peroxidase (HRP), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase, glutathione reductase and their enzymatic mimetics. Alternatively, the enzymes may be enzymes involved in biochemical enzymatic cascades, which refers to a series of biochemical reactions involving enzymes, such as blood coagulation, metabolism pathways, and signal transduction pathways. HSN may be synthesized by silica sol-gel process of water-in-oil microemulsion, and polyethyleneimine (PEI) modified enzymes in aqueous phase are then encapsulated inside HSN. It has been demonstrated that encapsulation of HRP in the cavities of HSN (HRP@HSN) is feasible and the intracellular delivery of HRP@HSN showing its function as a catalytic nanoreactor inside Hela cells for converting prodrug into toxic agent to kill the cancer cells.

In one embodiment, PEI grafted superoxide dismutase (PEI-SOD) and catalase (PEI-CAT) are prepared and then co-encapsulated within HSN as the loaded enzymes.

Synthesis of Enzyme-Encapsulated HSN

Herein, polyethyleneimine (PEI) is introduced to improve the enzyme loading. PEI, with rich amine groups, has been demonstrated to be good proton sponge for endosome escape effect. FIGS. 1A-1B describe the reaction schemes for the PEI-modification of the enzymes (SOD and CAT) and the encapsulation of the positively charged PEI-SOD and PEI-CAT within HSN. The silica shell of HSN shields off PEI from contacting with cellular machineries, but the pores of the silica shells allow protons to diffuse inside the hollow spheres of HSN and keep the proton sponge effects of PEI.

In FIG. 1A, the PEI-enzyme conjugation is achieved through the amidation of the carboxyl group with PEI. FIG. 1B illustrates the synthesis of PEI-SOD and PEI-CAT co-encapsulated HSN (denoted as PEI-SOD/CAT@HSN).

Both SOD and CAT are important antioxidant enzymes in living systems. PEI is covalently linked to the enzymes by the conventional EDC/NHS coupling reaction. Subsequently, the PEI-grafted enzymes (PEI-SOD, PEI-CAT), either individually or together, are encapsulated inside HSN by an one-pot water-in-oil (w/o) microemulsion approach as shown in FIG. 1B. In brief, an aqueous solution containing PEI-grafted enzyme is emulsified in an oil system containing surfactant (isooctylphenyl ether, CA-520), co-surfactant (n-hexanol) and organic solvent (decane). After introducing the silica sources, tetraethoxysilane (TEOS) and aminopropyltrimethoxy silane (APTMS), and aqueous ammonia, the water-in-oil (w/o) mixture was stirred for 10 h at 20° C. to form enzyme-encapsulated silica nanoparticles. Enzymes-loaded particles were foiiiied after the nanoparticles were further suspended in waiin deionized water (40° C.) for 40 min and washed with water, which is critical for the transformation to hollow nanospheres.

Modification of SOD and CAT with PEI: PEI-conjugation of enzymes was carried using NHS/EDC crosslinking reaction. Briefly, 3 mg of SOD and CAT were dissolved in 50 mM sodium phosphate buffer (pH 7.8) at a concentration of 3 mg/mL. Amine-reactive NHS esters and EDC·HCl were prepared in 200 μL sodium phosphate buffer (50 mM, pH 7.8) and then added to the enzyme solution. The SOD solution contained 17 mg NHS/14 mg EDC·HCl and CAT solution contained 15 mg NHS/13 mg EDC·HCl. After stirring for 1 h at 4° C., 43 and 38 μL of PEI reagent were added to the SOD and CAT solutions separately and the mixtures were further stirred at 4° C. for 24 h to complete the reactions. Finally, the PEI-conjugated SOD (PEI-SOD) and CAT (PEI-CAT) were purified and concentrated with Amicon Ultra filters (MA, USA).

Synthesis of PEI-SOD@HSN, PEI-CAT@HSN, and PEI-SOD/CAT@HSN: Hollow silica nanospheres (HSN) were synthesized by a reverse microemulsion method. Typically, 20 mL of decane, 1.63 mL of CA-520, 550 μL of n-hexanol and 350 μL of H₂O with concentrated PEI-enzymes (PEI-SOD, PEI-CAT, or a mixture of PEI-SOD and PEI-CAT) were mixed at room temperature to generate the water-in-oil microemulsion. Then, silica sources (100 μL of TEOS and 25 μL of ethanolic APTMS solution) were added with stirring. The ethanolic APTMS solution was prepared by adding 200 mL of APTMS to 1.4 mL of absolute ethanol. After 10 min, 250 μL of aqueous ammonia (35 wt %) was introduced to the mixture which was stirred for 10 h at 20° C. Then 95% ethanol was added to destabilize the microemulsion system and solid products were centrifuged at 11000 rpm for 30 min. To obtain the hollow structure, the samples were suspended in 40 mL warm water (40° C.), stirred for 40 minutes, and isolated by centrifugation to obtain PEI-SOD encapsulated HSN (denoted as PEI-SOD@HSN), PEI-CAT encapsulated HSN (denoted as PEI-CAT@HSN), PEI-SOD/CAT@HSN or HSN only (without adding enzymes). Finally, all nanoparticles were washed several times with ethanol and deionized water to extract the surfactants in the pores. The amount of PEI-enzymes used to prepare enzyme-loaded HSN: 36 nmole of PEI-SOD for PEI-SOD@HSN; 4 nmole of PEI-CAT for PEI-CAT@HSN; 36 nmole of PEI-SOD and 4 nmole of PEI-CAT for PEI-SOD/CAT@HSN.

Physicochemical Characterization.

The surface area of HSN (231.2 m²/g) as determined by N₂ adsorption—desorption isotherms exhibited characteristic type IV BET isotherms (FIG. 1). The high surface area is attributed to the porous structure on the shell, indicating most of the surfactants have been removed. FIGS. 3A-3D show transmission electron microscopy (TEM) images of enzyme encapsulated silica nanospheres; FIG. 3A: PEI-SOD/CAT@HSN, FIG. 3B: PEI-SOD/CAT@HSN stained with uranyl acetate (UA); FIG. 3C: PEI-SOD@HSN and FIG. 3D: PEI-CAT@HSN. All particles show a narrow size distribution, with an average diameter of about 40 nm. Uranyl acetate (UA) staining displayed an enhanced electron density inside PEI-SOD/CAT@HSN but no staining outside the particles (FIG. 3B). This indicates the enzymes were indeed entrapped within the hollow spheres.

The enzyme-encapsulation efficiency and loading yields have been examined and the results are listed in Table 1. Significantly higher amount of enzymes are encapsulated in HSN with the cationic polymer PEI as shown in Table 1.

TABLE 1 SOD-Loading CAT-Loading yield yield SOD-encapsulation CAT-encapsulation (μg (μg efficiency efficiency enzyme/mg enzyme/mg Sample (%) (%) particles) particles) SOD@HSN  2.8% (±0.2%) CAT@HSN 2.8% (±0.4%) PEI-SOD@HSN 12.2% (±3.1%) 44.7 (±8.1) PEI-CAT@HSN 8.7% (±2.3%) 29.5 (±4.6) PEI-SOD/CAT@HSN 12.3% (±3.4%) 9.4% (±0.8%) 44.5 (±5.7) 26.1 (±3.3)

The loading efficiency of PEI-SOD (12.2%) and PEI-CAT (8.7%) were much higher than that of SOD (2.8%) and CAT (2.8%). This is due to the positively charged PEI-SOD and PEI-CAT are effective in attracting the negatively charged hydrolyzed silica precursors for encapsulation. The higher loading will improve the enzyme activity for biomedical use and thus PEI-coated enzymes are chosen for further study thereafter. The loading yields of PEI-SOD@HSN and PEI-CAT@HSN were 44.7 and 29.5 μg enzymes/mg HSN. Compared with single enzyme loading, about the same level of efficiency and loading yields were observed when two enzymes, PEI-SOD and PEI-CAT, were co-encapsulated in HSN. The loading efficiency of PEI-SOD and PEI-CAT were 12.3% and 9.4%. PEI-SOD/CAT@HSN contains about 44.5 and 26.1 μg enzymes/mg HSN (Table 1).

Enzymatic Activity Assays

In the case of SOD, samples were prepared in 300 μL and monitored using a microplate reader (Bio Tek, Synergy™ H1). First, a stock of cocktail reagent contained EDTA (10-4 M), cytochrome c (10-5 M), and xanthine (5×10-5 M) in 1 mL of 50 mM K₃PO₄ was prepared. Then, 280 μL of cocktail reagent was added with various samples (native SOD, PEI-SOD/CAT@HSN, PEI-SOD@HSN, PEI-CAT@HSN or HSN). Then, xanthine oxidase (10 μL of 58 mU/mL) was added to the above solution completing with D.I. water up to 300 μL total volume. Finally, 200 μL of each sample was transferred to microplate reader and the absorbance at 550 nm was determined. To measure the SOD activity, the inhibition rate of cytochrome c reduction between native SOD (3000 U/mg) and SOD loaded particles (PEI-SOD/CAT@HSN and PEI-SOD@HSN) were compared using the slopes of absorbance between t=0 sec and t=180 sec.

In the case of CAT assay, 90 μL of H₂O₂ solution (5 μM in 50 mM sodium phosphate buffer; pH 7.8) and various nanoparticles solutions (native CAT, PEI-SOD/CAT@HSN, PEI-CAT@HSN, PEI-SOD@HSN or HSN) were mixed and completed with D.I. water up to 100 μL total volume. After incubation for 30 min, HRP (100 μL of 2 U/mL) and Amplex red (10 μL of 0.1 mM) were added. Each sample (200 μL) was transferred to a microplate reader and the absorbance was monitored at 570 nm. To determine the CAT activity, we calculated the ΔOD₅₇₀ of each sample between t=0 min and t=30 min and compared with native CAT. Table 2 shows the results of relative activity of SOD and CAT after encapsulated inside HSN.

TABLE 2 Relative activity Relative activity Sample (SOD) (CAT) Native SOD 100 PEI-SOD@HSN 18.6 (±13.4) Native CAT 100 PEI-CAT@HSN 62.7 (±29.2) PEI-SOD/CAT@HSN 46.6 (±5.8)  57.9 (±30.5)

Compared with the inhibition percentage of cytochrome c reduction between native SOD and SOD-loaded particles, the encapsulated PEI-SOD/CAT@HSN and PEI-SOD@HSN maintain 46.6% and 18.6% of the native SOD activity (Table 2). Also, as seen in Table 2, when compared with the reaction rate of H₂O₂ decomposition between native CAT and CAT-loaded particles, PEI-SOD/CAT@HSN and CAT@HSN keep 57.9% and 62.7% of the native CAT activity.

The synergism between SOD and CAT in the presence of O₂ ^(•−) was investigated. FIG. 4A shows the stability of CAT-loaded particles and native CAT after treated with KO₂ solution. As shown in FIG. 4A, after exposure to KO₂ solution, which generates 0₂ ^(•−), native CAT lost almost all its activity, whereas CAT activity of PEI-SOD/CAT@HSN and PEI-CAT@HSN still remained at about 80% and 70%.

Cascade reactions in PEI-SOD/CAT@HSN

SOD and CAT are antioxidant enzymes and work together as primary defence system against free radical damage in living cells. In aforementioned study, we had demonstrated that PEI-SOD and PEI-CAT could be simultaneously encapsulated in HSN and the final nanoparticle system showed activities in both enzymes. In order to investigate whether both the encapsulated enzymes are involved in cascade reactions, fluorescence assay was performed in which the resorufin formation was recorded by monitoring the fluorescence at 570 nm. In the reaction, the O₂ ^(•−) generated by KO₂ was unstable and converted to H₂O₂ in the presence or absence of SOD in aqueous solution. However, the dismutation rate of O₂ ^(•−) in the presence of SOD is faster. Subsequently H₂O₂ could be either reduced by CAT or further involved in peroxidase/Amplex red oxidation reaction. As shown in FIG. 4(B), the fluorescence intensity of PEI-SOD@HSN was significantly higher than that of blank (control), HSN (negative control), and PEI-SOD/CAT@HSN. The results indicated PEI-SOD@HSN catalyzed the reduction of O₂ ^(•−) to hydrogen peroxide and increase the rate of resorufin formation. However, in the presence of PEI-SOD/CAT@HSN, the cascade reaction occurs. Hydrogen peroxide produced by SOD could be further decomposed by CAT and the system showed weaker fluorescence. Relative enzyme activity was expressed as a ratio of change in absorption at 570 nm before and after KO₂ treatment. FIG. 4B shows the cascade reactions within PEI-SOD/CAT@HSN. The fluorescence intensity reflects the amount of H₂O₂ in the assay buffer. Herein, the same SOD units of PEI-SOD/CAT@HSN and PEI-SOD@HSN were used for comparison.

Cell proliferation assay: 2×10⁴ cells were seeded in 24-well plates and allowed to attach for 24 h. To determine the particle toxicity, cells were incubated in fresh serum-free medium containing different amounts of BSA-stabilized particles (0-400 μg/mL) for 2.5 h. After washing twice with phosphate-buffered saline (PBS), particle-treated cells were cultured in regular growth medium. After 24 h, cells were washed twice with PBS and incubated with 200 μL WST-1 (10%) in Dulbecco's modified eagles medium (DMEM). Cell viability was determined by the fornazan dye generated by the live cells and the absorbance at 450 nm was measured using a microplate reader (Bio-Rad, model 680).

Cell Uptake, Distribution Pattern, and Cytotoxicity of PEI-Enzyme Loaded HSN

From the experimental observations, nanoparticles, HSN and PEI-SOD/CAT@HSN are well-internalized into cells, and most of the nanoparticles have escaped from endosome and resided in the cytoplasm. The cell uptake of the nanoparticles, at the treated doses of 50, 100 and 200 μg/mL for 2.5 h, leads to remarkably high cellular uptake of PEI-SOD/CAT@HSN and a dose-dependent uptake behaviour of nanoparticles by HeLa cells. FIG. 5 shows the cell viability results for Hela cells exposed to HSN or PEI-SOD/CAT@HSN using WST-1 assay. More importantly, no significant cytotoxicity of HSN and PEI-SOD@HSN are found in cell proliferation assay while the doses of particles were up to 400 μg/mL (FIG. 5). Cell viability was examined using WST-1 assay at doses of 25, 50, 100, 200, and 400 μg/mL, after 2.5 h of particle treatment and followed by additional 24 h growth.

Protective Effect of Enzyme-Loaded Particles in Cells after Paraquat (PQ) Treatment

It has been demonstrated that tandem reaction occurs within PEI-SOD/CAT@HSN in test tube experiments. The nanoparticles exhibited excellent synergism effect between SOD and CAT. They also displayed high bio-compatibility. These advantages provided opportunities for their uses in protein therapy. The capability of enzyme-loaded particles in living cells for removing paraquat (PQ)-induced ROS is evaluated. ROS production is measured by flow cytometry with dihydroethidium (DHE), a superoxide indicator with fluorescence upon oxidation, to evaluate the antioxidant effect of each kind of nanoparticle. HeLa cells were treated with various nanoparticles at a dose of 100 μg/mL for 2.5 h. After washing twice with PBS, cells were exposed to 500 μM of paraquat (PQ) for 18 h and then stained with 2 μM of dihydroethidine (DHE) and the oxidized DHE with fluorescence was determined by flow cytometry analysis. FIG. 6 shows the quantification of fluorescence intensity of oxidized DHE from HeLa cells were treated with various nanoparticles. The PQ-induced ROS productions are the same for the un-protected cells (positive control and HSN only). All enzyme-nanoparticle treated cells obviously inhibit PQ-induced ROS production. PEI-SOD@HSN and PEI-CAT@HSN show similar levels of reduction of DHE-positive cells by about 25% (compared to HSN only). However, co-loaded PEI-SOD/CAT@HSN shows a much higher level of reduction of DHE-positive cells by about 60%. The PEI-SOD/CAT@HSN nanosystem clearly shows a synergetic effect in ROS reduction. DHE-positive cells and fluorescence intensity quantitated indicates that PEI-SOD/CAT@HSN having the synergetic effect of both antioxidant enzymes displays the weakest fluorescence intensity under microscopic observation. The enzyme-loaded particles, especially PEI-SOD/CAT@HSN, show better cell attaching morphology, which implies the treatment by PEI-SOD/CAT@HSN detoxify the imposed ROS. The nanoparticle-treated cells after incubation with PQ were further assayed with Western blotting to check the level of p-p38 and COX2 expression. Down-regulation of PQ-induced activation of p-p38 and COX2 is observed for cells pre-treated with PEI-SOD/CAT@HSN, PEI-SOD@HSN and PEI-CAT@HSN.

In one aspect, the present invention provides a mesoporous hollow silica nanosphere (HSN) materials (about 40 nm size) based on templating water-in-oil with sol-gel condensation. The enzyme SOD and/or catalase (CAT) can be individually or jointly encapsulated within HSN, with the help of the polyamine PEI. The mesopores on the silica shell allow easy access of small molecules while keeping the enzymes inside the nanosphere from undesirable protein-protein interaction. We found excellent catalytic activities of superoxide dismutation by SOD and then transformation of H₂O₂ to water by catalase encapsulated in HSN. When PEI-SOD and PEI-CAT are co-encapsulated, PEI-SOD/CAT@HSN can complete cascade transformation of superoxide through hydrogen peroxide to water with synergism.

After delivery to cells, substantial fractions of HSN exhibited endosome escape to cytosol. Downstream reactive oxygen species (ROS) production and COX-2/p-p38 expressions show that co-encapsulated SOD/CAT inside HSN give the highest cell protection against the toxicants. The rapid cell-uptake and strong detoxification effect on superoxide radicals by the SOD/CAT-encapsulated hollow mesoporous silica nanoparticles demonstrate that the enzyme-encapsulated hollow mesoporous silica nanoparticles can function as a catalytic nanoreactor after cell uptake. PEI-SOD/CAT@HSN may be applied as antioxidants for medical treatments, such as inflammation, ischemia, stroke and other strong oxidative stress situation.

However, the sizes of many proteins or enzymes are significantly bigger than the inner space of HSN or MSN or certain proteins are not suitable to be encapsulated within the nanomaterials and the loading efficiency is limited. In this case, it is feasible to link or conjugate the enzymes or proteins to the exterior or outer surface of the nanoparticles or nanomaterials for better delivery efficiency.

Alternatively, in another embodiment, mesoporous silica nanoparticle (MSN) materials are synthesized and functionalized to carry peptides and/or antibodies. The peptide may be any peptide containing cysteine or a polyhistidine tag, including nucleus localization sequence (NLS)-peptide, cancer-targeting peptide and lysosomal targeting peptide. The antibody may be any antibody containing cysteine or a polyhistidine tag, including signal transduction antibody and cancer-targeting antibody.

The present invention provides nanoparticles consisting of mesoporous silica nanoparticle (MSN) with surface functionalization of NF-κB (nuclear factor-kappa B) p65 antibody and TAT transducing peptide (i.e., HIV trans-activator of transcription (TAT) protein transduction domain). The sequence of TAT transducing peptide: CGRKKRRQRRR. These nanoparticles can move near nuclear membrane and block nuclear translocation of the activated p65.

FIG. 7 describes the reaction scheme for the conjugation of NF-κB p65 antibody and Cys-TAT peptide to the surface functionalized MSN. To synthesize the functionalized MSN, amine groups are formed on the surface of MSN by reacting with 3-aminopropyltrimethoxysilane (APTMS) to foim MSN-APTMS with an average loading of nitrogen content of APTMS at 2.6 wt % by elemental analysis. In order to immobilize p65 antibody on MSN, we chose two polyethylene glycol (PEG) crosslinkers with different lengths, MAL-PEG_(2k)-SCM and MAL-PEG_(3.4k)-SCM, to MSN-APTMS. Herein, the abbreviations are explained, MAL: maleimide, PEG_(2k) or PEG_(3.4k): polyethylene glycol (PEG) having an average molecular weight of 2000 or 3400, SCM: succinimidyl carboxymethyl. The MAL-PEG-SCM crosslinkers containing a succinimidyl moiety react with the amine groups of MSN-APTMS through an active succinimidyl link to obtain the MSN-PEGs (MSN-PEGs: MSN-PEG_(2k), MSN-PEG_(34k)). The MAL-end of MSN-PEG reacted with the thiol groups of the antibody and Cys-TAT peptide.

Synthesis of Green Fluorescent Mesoporous Silica Nanoparticles (MSN) C₁₆TABr (0.58 g, 1.64×10⁻³ mole) and 5 mL of 0.226 M ethanol solution of tetraethoxysilane (TEOS, 1 mL TEOS in 20 mL 99.5% ethanol) were dissolved in 300 g of 0.17 M aqueous ammonia solution. The stock solution was stirred at 40° C. for 5 h. 5 mL of 1.13 M ethanol solution of TEOS (5 mL of TEOS in 20 mL 99.5% ethanol) and FITC-APTMS were added with vigorous stirring for 1 h and then aged statically at 40° C. for 24 h. FITC-APTMS, N-1-(3-trimethoxysilyl propyl)-N′-fluoreceylthiourea), was prepared in advance by stirring fluorescein isothiocyanate (FITC, 1 mg) and 3-aminopropyltrimethoxysilane (APTMS, 100 μL) in 5 mL ethanol (99%) at room temperature for 24 h. As synthesized samples were then collected by centrifugation with 12000 rpm for 20 min and washed five times with 99% ethanol. 200 mg of as-synthesized samples were redispersed in 25 mL of 95% ethanol with 0.5 g of 37 wt % HCl. Surfactant was extracted by heating the ethanol suspension at 60° C. for 24 h. The product, called FITC-MSN (MSN), was collected by centrifugation and washed with ethanol several times and stored in ethanol.

Preparation of Amine-Functionalized MSN (MSN-APTMS)

The surface of MSN was functionalized with amine groups by treatment with APTMS. MSNs (200 mg) were first dispersed in 50 mL of ethanol, and then the solution was refluxed for 18 h after the addition of 500 μL of APTMS. After centrifugation and washing with ethanol, amine-functionalized MSNs were redispersed in ethanol. To remove the surfactants, the amine-functionalized MSNs were suspended in acidic ethanol (1 g of HCl in 50 mL of EtOH) and refluxed for 24 h. After centrifugation and washing with ethanol, amine-functionalized MSN (MSN-APTMS) were redispersed in ethanol.

Transmission Electron Microscopy (TEM)

TEM images were taken using a Hitachi H-7100 instrument with an operating voltage of 75 KV. Samples were sonicated to disperse in ethanol and 10 μL of the suspension was dropped to fix on a microgrid.

For evaluation of immunological efficiency, the p65 antibody was covalently immobilized with the MAL-end of MSN-PEG_(3.4k) in different ratios (1:6, 1:12, 1:24) via C-S binding. After the p65 antibody conjugation, the Cys-TAT peptide was conjugated to fill up the free MAL-end of MSN-PEG_(3.4k). MSN-PEG_(3.4k) without antibody coupling was also directly conjugated with Cys-TAT as a control. The physical properties of the nanoparticles were characterized by nitrogen adsorption-desorption isotherms, powder X-ray diffraction (XRD), FT-IR, TEM, dynamic light scattering (DLS) and zeta potential. FIGS. 8A-8D show transmission electron microscopy (TEM) images of various functionalized MSN. From the TEM images, it is shown that the MSN particles possess well-ordered mesoporous structures and the average particle size obtained from TEM images is about 40 nm.

Cell Viability and Growth Inhibition Assay.

The WST-1 assay was applied to measure the cell viability and growth inhibition assay: 2×10⁴ HeLa cells per well were seeded in 24-well plates for 16 h for HeLa cell viability assay. HeLa cells were incubated in serum-free medium containing different amounts of MSN-PEG_(3.4k)-Ab-TAT (100 μg/L) for 4 h. For HNSCC growth inhibition assay, HNSCC cells were seeded in 24-well plates with a density of 4×10⁴ cells/well for 16 h and incubated with 200 μg/mL of MSN-PEG_(3.4k)-Ab(1:24)-TAT, MSN-PEG_(3.4k)-TAT or anti-TNF antibody (100 ng/mL) in serum-free medium for 4 h. Following medium replacement with culture medium, HNSCC cells were incubated for further 72 h. For WST-1 assay, HeLa or HNSCC cells were allowed to grow in culture medium containing WST-1 (Clontech) for 4 h at 37° C. The dark red formazan dye generated by the live cells was proportional to the number of live cells and the absorbance at 450 nm was measured using a microplate reader (Bio-Rad, model 680).

The cell viability of the MSN-PEG_(34k)-Ab-TAT was examined by using WST-1 assay and MSN-PEG_(3.4k)-Ab-TAT shows no significant cytotoxicity.

Western Blotting Analysis

Cell lysates were separated on a 10% SDS-PAGE, and the proteins were then electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane and blocked 1 h at room temperature in blocking buffer [1×Tris-buffered saline (TBS)-0.1% Tween 20, 5% w/v nonfat dry milk]. Membranes were incubated overnight at 4° C. with primary antibodies: NF-κB p65, TNF-α, Lamin B and GAPDH from Santa Cruz Biotechnology (Santa Cruz, Calif.), along with COX-2 from Cayman (Cayman, Ann Arbor, Mich., USA). All primary antibodies were diluted in blocking buffer (NF-κB p65: 1:500, TNF-α: 1:500, Lamin B: 1:3500, GAPDH: 1:5000 and COX-2: 1:500 dilution). The PVDF membranes were extensively washed and incubated with a horseradish peroxidase—conjugated secondary immunoglobulin G antibody (1: 2000 dilution, Santa Cruz Biotechnology) for 1 h at room temperature. Immunoreactive bands were visualized with an enhanced chemiluminescence substrate kit (Amersham Pharmacia Biotech, GE Healthcare UK Ltd, Bucks, UK) according to the manufacturer's protocol.

Cellular Response of NF-κB on MSN-PEGs and in vitro Pull-Down Assay of MSN-PEG_(3.4k)-Ab-TAT

100 μg/mL of MSN-APTMS, MSN-PEG_(2k) and MSN-PEG_(3.4k) were treated in HeLa cells for 4 h, and then incubation without or with TNF-α (50 ng/mL), a NF-κB activator, for another 0.5 h. After the cells were harvested, cytosolic and nuclear protein were isolated, the p65 expression level in either cytosol or nucleus was determined by western blotting experiments. For in vitro pull-down assay, the MSN-PEG_(3.4k)-Ab-TAT (100 μg/mL) was mixed and incubated with total lysate of HeLa cell at 4° C. for 18 h in vitro. Then, the mixture was centrifuged at 12,000 rpm for 20 mins and the supernatant (10 μL) was assayed for the free p65 expression level by Western blotting.

FIGS. 9A-9C show the results of in vitro pull-down assay of various functionalized MSN nanoparticles. MSN-PEG_(3.4k)-Ab-TAT blocks NF-κB p65 nuclear translocation and thus inhibits the NF-κB p65 downstream protein expression. HeLa cells were treated with MSN-PEG_(3.4k)-TAT or MSN-PEG_(3.4k)-Ab-TAT for 4 h at different doses (100 μug/mL for FIG. 9B, 50-200 μg/mL for FIG. 9C). After the delivery, the cells were stimulated with or without 5Ong/mL TNF-α for another 0.5 h. Dose-dependence study of the blockage as shown in FIG. 9C indicates that nuclear p65 level decreases with the increasing concentration of MSN-PEG_(3.4k)-Ab(1:24)-TAT.

As shown in FIG. 9B, western blotting was carried out to quantify the p65 level in HeLa cells. After the treatment, the cell lysates were harvested for the p65 level in nucleus and cytoplasm. The western blotting results indicated that MSN-PEG_(3.4k)-Ab-TATs did not induce any nuclear translocation of p65 without TNF-α. However, under the TNF-α treatment, a significant increase of p65 level appeared in the nucleus in absence of MSN-PEG_(3.4k)-Ab-TATs. Once the MSN-PEG_(34k)-Ab-TATs with different amount of conjugated antibody were added, the level of nuclear p65 gradually reduced with increasing amount of antibody. Both MSN-PEG_(3.4k)-Ab(1:12)-TAT and MSN-PEG_(3.4k)-Ab(1:24)-TAT show obvious suppression of the p65 translocation to nucleus, whereas MSN-PEG_(3.4k)-TAT did not prevent the TNF-α inducing nuclear p65 translocation. Hence, MSN-PEG_(3.4k)-Ab-TAT shows the specificity and effectiveness to block NF-κB p65 nuclear translocation through immunogenic binding.

Herein, a nanoparticle/antibody complex targeting NF-κB is employed to catch the Rel protein p65 in perinuclear region and thus blocking the translocation near the nuclear pore gate. TAT peptide conjugated on mesoporous silica nanoparticles (MSN) help non-endocytosis cell-membrane transducing and converge toward perinuclear region, where the p65 specific antibody performed the targeting and catching against active NF-κB p65 effectively.

In another embodiment, a protein delivery system combining MSN nanoparticle carriers and one or more denatured fusion proteins has been developed. Such combination of the nanomaterial and one or more fusion proteins not only solves the problems of protein delivery, including chemical solvents, stability, and permeability, but also provide a new approach for protein therapy.

Herein, two antioxidant enzyme proteins with similar function for free radicals scavenging, superoxide dismutase (SOD) and glutathione peroxidase (GPx), are demonstrated as the co-delivered enzymes carried by the nanoparticles.

For TAT-SOD and TAT-GPX protein conjugation, we constructed and overexpressed the His-tag human Cu, Zn-superoxide dismutase (SOD) and human glutathione peroxidase (GPx) which contain a human immunodeficiency virus (HIV) transducing domain (TAT, residues 49-57). The sequence of TAT transducing peptide: RKKRRQRRR. The genes of TAT-SOD and TAT-GPx were cloned and inserted into prokaryotic protein expression vector of pQE-30 to form pQE-TAT-SOD and pQE-TAT-GPx. The vectors were transformed into JM109 E. coli and cultured in LB broth with IPTG protein induction for 1 and 3 h. The TAT-SOD and TAT-GPx with high protein overexpression were displayed in accordance with increasing induction time in 10% SDS-PAGE electrophoresis. Finally, the supernatants of pellets of E. coli crude lysates expressed TAT-SOD or TAT-GPx were tried to further directly conjugate in 8M urea.

Synthesis of Fluorescent Mesoporous Silica Nanoparticles (FMSN)

Dye-functionalized MSNs were synthesized by co-condensation process. FITC solution was prepared by dissolving 1 mg of FITC in 5 ml of anhydrous ethanol. 100 L of APTMS was added with rapid stirring at room temperature in darkness for 24 hours. 0.58 g of C₁₆TAB was dissolved in 300 g of 0.17 M NH₃ solution, and 5 mL of dilute TEOS solution (5% v/v TEOS/ethanol) was added with stirring for 5 h. FITC-APTMS solution added before 5 ml of concentrate TEOS solution (25% v/v TEOS/ethanol) was added dropwise with vigorous stirring for 1 h. The solution was then aged at 40° C. for 24 hours to complete the silica condensation. As-synthesized products was collected by centrifugation and washed with 95% ethanol three times. The products called FITC-MSN (FMSN) were stored in absolute ethanol.

Conjugation of NTA-Silane and Ni (II) with FMSN

100 mg of FMSN were suspended in 50 mL of toluene containing 50 mg of NTA-silane and reflux for 18 h. The products were cleaned by ethanol to eliminate excess silane. In order to remove the C₁₆TABr templates, the particles were dispersed in acidic solution (1 g of HCl in 50 mL ethanol) and stirred at 60° C. for 24 h. Subsequently, hydrolysis of methoxycarbonyl on NTA linker was achieved in the presence of aqueous p-TsOH (0.266 g, pH=2.0) under stirring at 65° C. for 6 h. After cleaned by ethanol, the particles were reacted with 50 mM NiCl₂ aqueous solution for 6 h at room temperature. Followed the same cleaned procedure described above, the

FMSN-NTA-Ni were obtained and stored in absolute ethanol. FMSN-NTA-Ni with an average loading of Ni content is 0.6 wt % by ICP-MS analysis.

Synthesis of FMSN-PEG/PEI Nanoparticles

Dye-functionalized MSNs were synthesized by co-condensation process. FITC solution was prepared by dissolving 1 mg of FITC in 5 mL of anhydrous ethanol. 100 of APTMS was added with rapid stirring at room temperature in darkness for 24 hours. 0.58 g C₁₆TAB was dissolved in 300 g of 0.17 M NH₃ solution, and 5 mL of dilute TEOS solution (5% v/v TEOS/ethanol) was added with stirring for 5 h. FITC-APTMS solution added before 5 mL of concentrate TEOS solution (25% v/v TEOS/ethanol) was added dropwise with vigorous stirring for 1 h. 900 μL PEG-silane and 40 μL PEI-silane were added and stirring for 30 mins. The solution was then aged at 40° C. for 24 hours to complete the silica condensation. The solution was aged under hydrothermal condition at 90° C. for 24 hours and 70° C. for 24 hours. As-synthesized products was collected by centrifugation and washed with 95% ethanol. The particle was redispersed in 50 mL of 95% ethanol with 1 g of 37 wt % HCl for 1 hour and then the acid solvent was changed to 50 mL of 95% ethanol with 50 μL of 37 wt % HCl to remove the CTAB. FITC conjugated MSN (FMSN)-PEG/PEI _(p)articles were collected by centrifugation and washed with 95% ethanol three times.

Characterization

Transmission electron microscopy (TEM) images were taken on a JEOL JSM-1200 EX II operating at 120 kV. The nickel amount of sample was determined by inductively coupled plasma mass spectrometry (ICP-MS) using Agilent 7700e instrument. Size measurements were performed using dynamic light scattering (DLS) on a Malvern Zetasizer Nano ZS (Malvern, UK). Zeta potential was determined by the electrophoretic mobility and then applying the Henry equation on Malven Zetasizer Nano ZS (Malvern, UK). Table 3 shows dynamic light scattering (DLS) data for average particle size of FMSN-PEG/PEI nanoparticles in different solutions.

TABLE 3 solvent Size (nm) H₂O 65.04 ± 0.57 PBS 63.03 ± 0.34 DMEM 63.71 ± 0.80 DMEM + FBS 69.41 ± 0.40

FIG. 10A shows the conjugation of FMSN-PEG/PEI nanoparticles, while FIG. 10B shows the TEM images of FMSN-PEG/PEI nanoparticles. The TEM images show that these FMSN-PEG/PEI particles possess well-ordered mesoporous structure with an average particle size of about 60-70 nm. DLS-determined size indicates very little aggregation in biological solutions (Table 3).

Conjugation of NTA and Ni (II) with FMSN-PEG/PEI

20 mg of FMSN-PEG/PEI was dispersed in 2.5 mL of PBS buffer, and then 6.8 mg of NHS-PEG-MAL(3.4k) was dissolved in 2.5 mL of PBS and then added to FMSN-PEG/PEI solution. The solution was stirred for 2 hours at room temperature. Thiolated Na,Na-Bis(carboxymethyl)-L-lysine hydrate (BCLH) solution was prepared by added 400 μL of Traut's reagent (100 μM) and 5.24 mg of Na,Na-Bis(carboxymethyl)-L-lysine hydrate in 5 mL of PBS buffer and stirred for 30 mins. The thiolated BCLH solution was added to the FMSN-PEG/PEI solution and stirred overnight at 4° C. . Subsequently, hydrolysis of methoxycarbonyl on NTA linker was achieved in the presence of aqueous p-TsOH (0.133 g, pH=2) under stirring at 65° C. for 6 h. After washed by ethanol, the particles were reacted with 50 mM of NiCl₂ aqueous for 6 h at room temperature. Followed the same washed procedure described above, the FMSN-PEG/PEI-NTA-Ni were obtained and stored in absolute ethanol.

Immobilization of His-TAT-Protein with FMSN-NTA-Ni

The lysate of E.coli containing His-TAT-SOD or His-TAT-GPx was mixed with FMSN-NTA-Ni at 4° C. overnight. Based on the metal affinity between the Ni (II) and His-tag protein offered a tight linkage with a very low dissociation constant, the FMSN-NTA-Ni was directly mixed with TAT-SOD or TAT-GPx proteins from the supernatants of pellets of E. coli crude lysates under 8M urea without purifying.The protein-conjugated particles were isolated by centrifugation and washed by ethanol. The protein-functionalized particles were denoted as FMSN-TAT-SOD or FMSN-TAT-GPx.

Determination of SOD and GPx Activity

In the case of SOD, samples were prepared in 300 μL and monitored using a microplate reader (Bio Tek, Synergy™ H1). Firstly, a stock of cocktail reagents contained EDTA (10⁻⁴ M), cytochrome c (10⁻⁵ M), and xanthine (5×10⁻⁵ M) in 1 mL of 50 mM K₃PO₄ was prepared. Then, 280 μL of cocktail reagent was added with various samples, xanthine oxidase (10 μL of 58 mU/mL) and completed with D.I. water up to 300 μL total volume. Finally, 200 μL of each sample was transferred to microplate reader and the absorbance at 550 nm was detection. To measure the SOD activity, the inhibition rate of cytochrome c reduction between native SOD and SOD samples were compared using the slopes of absorbance between t=0 sec and t=180 sec. SOD specific activity was expressed as unit per milligram (U/mg) of total lysate proteins (The Journal of Biological Chemistry, 1969, 244, 6049-6055.).

GPx activity in HeLa cell was measured using the Glutathione Peroxidase Assay Kit (Cayman Chemical), based on the method of Paglia and Valentine, with hydrogen peroxide as substrate. The method was based on an NADPH-coupled reaction, whereby GPx reduces hydrogen peroxide while oxidizing GSH to GSSG. The generated GSSG is reduced to GSH with consumption of NADPH by GR. Enzyme activity was measured at 340 nm and expressed in units representing oxidation of 1 μmole NADPH per minute per mL sample. GPX specific activity was expressed as unit per milligram (U/mg) of protein.

Cell Viability Assay: 3×10⁴ cells per well were seeded in 24-well plates for proliferation assays. After incubation with different amounts of nanoparticles suspended in serum-free medium for 4 h, respectively, then the 500 μM N, N′-dimethyl-4, 4′-bipyridinium dichloride (paraquat) was added to the culture medium for 24 h. Particle-treated cells were then washed twice with PBS and incubated with 200 μL WST-1 (10%) in DMEM. Cells viability was estimated by a formazan dye generated by the live cells and the absorbance at 450 nm was measured using a microplate reader (Bio-Rad, model 680).

FIGS. 11A-C show the protection effects of co-delivery of TAT-SOD and TAT-GPx into Hela cells. FIG. 11A shows the enhanced cell viability results for various nanoparticles by using WST-1 assay. FIG. 3B shows the results of ROS detection for various nanoparticles. The levels of ROS were stained by DHE assays and quantified by flow cytometry. FIG. 11C shows the results of Western blotting assays to show the levels of COX II and p-p38. The concentration of PQ and co-delivery of FMSN-TAT-SOD and FMSN-TAT-GPx (1:1 ratio) are 500 μM and 25 μg/mL, respectively.

Herein, it is shown that the denatured TAT-SOD or TAT-GPx fusion protein can be co-delivered into Hela cells and the denatured fusion proteins can be refolded and exhibit the specific enzymatic activities after delivering into the cells. Based on the results shown herein, the TAT-SOD or TAT-GPx fusion protein functionalized FMSN, named as FMSN-TAT-SOD or FMSN-TAT-GPx, still has the enzymatic activity by the refolding mechanism of the cells.

In conclusion, by using HSN or MSN, the mesoporous carriers of the present disclosure embodiments can deliver peptides, proteins, enzymes or enzymatic mimetics into the cells as needed and the native activities of the peptides, proteins, enzymes or enzymatic mimetics being delivered into the cell are maintained. The mesoporous carriers can function as nanoreactors located within the cells and the delivered peptides, proteins, enzymes or enzymatic mimetics can work together to provide multiple functions.

It will be apparent to those skilled in the art that various modifications and variations can be made to the structure of the present invention without departing from the scope or spirit of the invention. In view of the foregoing, it is intended that the present invention cover modifications and variations of this invention provided they fall within the scope of the following claims and their equivalents. 

What is claimed is:
 1. Mesoporous carriers, for delivering targets into a cell, comprising: hollow silica nanospheres (HSN) or mesoporous silica nanoparticles (MSN); and the targets carried by the hollow silica nanospheres or the mesoporous silica nanoparticles, wherein the targets at least includes first targets and second targets, and the first and second targets are different.
 2. The carriers of claim 1, wherein the mesoporous silica nanoparticles are surface-functionalized mesoporous silica nanoparticles functionalized with 3-aminopropyl-trimethoxysilane (APTMS).
 3. The carriers of claim 2, wherein the surface-functionalized mesoporous silica nanoparticles are bound with crosslinkers containing polyethylene glycol.
 4. The carriers of claim 3, wherein the first target is an antibody and the second target is a peptide respectively bound to the crosslinkers on surface-functionalized mesoporous silica nanoparticles.
 5. The carriers of claim 4, wherein the antibody is specific to transcriptional factors, mediators or complex in a signalling pathway and the peptide is a nucleus localization sequence (NLS)-peptide or a NLS like-peptide, so that the surface-functionalized mesoporous silica nanoparticles are sequestered outside of a nuclear membrane of the cell.
 6. The carriers of claim 4, wherein the antibody is NF-κB p65 antibody and the peptide is TAT transducing peptide.
 7. The carriers of claim 2, wherein the first target is superoxide dismutase and the second target is glutathione peroxidase respectively bound to the crosslinkers on surface-functionalized mesoporous silica nanoparticles.
 8. The carriers of claim 7, wherein the first target is TAT transducing peptide bound superoxide dismutase and the second target is TAT transducing peptide bound glutathione peroxidase respectively bound to the crosslinkers on surface-functionalized mesoporous silica nanoparticles.
 9. The carriers of claim 7, wherein at least one of superoxide dismutase and glutathione peroxidase is bound to the mesoporous silica nanoparticles in a denatured form or a partially active form.
 10. The carriers of claim 2, wherein the surface-functionalized mesoporous silica nanoparticles are labelled with a tracking agent or a dye.
 11. The carriers of claim 10, wherein the dye is fluorescein isothiocyanate.
 12. The carriers of claim 1, wherein the first and second targets are different enzymes or catalytic mimetics capable of catalysing a cascade reaction.
 13. The carriers of claim 12, wherein the enzymes or catalytic mimetics are capable of catalysing the cascade reaction involved in scavenges free radicles in the cell, so as to protect the cell against ROS induced stress.
 14. The carriers of claim 1, wherein at least one of the first and second targets is bound to the mesoporous silica nanoparticles in a denatured form or a partially active form.
 15. The carriers of claim 1, wherein the first and second targets are co-encapsulated within the hollow silica nanospheres.
 16. The carriers of claim 15, wherein the first and second targets are different enzymes or catalytic mimetics capable of catalysing a cascade reaction.
 17. The carriers of claim 16, wherein the enzymes or catalytic mimetics are capable of catalysing the cascade reaction involved in scavenges free radicles in the cell, so as to protect the cell against ROS induced stress.
 18. The carriers of claim 15, the first target is polyethyleneimine-grafted superoxide dismutase and the second target is polyethyleneimine-grafted catalase.
 19. A method of delivering targets into a cell, comprising: providing mesoporous carriers, wherein the mesoporous carriers comprises hollow silica nanospheres (HSN) or mesoporous silica nanoparticles (MSN) and the targets carried by the hollow silica nanospheres or the mesoporous silica nanoparticles, and the targets at least include first targets and second targets, and the first and second targets are different; contacting the mesoporous carriers with the cell by incubating the cell with the mesoporous carriers; and delivering the first targets and the second targets into the cell at the same time, wherein the mesoporous carriers and the targets carried by the hollow silica nanospheres or the mesoporous silica nanoparticles enter into the cell.
 20. The method of claim 19, wherein providing mesoporous carriers includes surface-functionalizing the mesoporous silica nanoparticles with 3-aminopropyl-trimethoxysilane (APTMS) to form surface-functionalized mesoporous silica nanoparticles.
 21. The method of claim 20, wherein the surface-functionalized mesoporous silica nanoparticles are bound with crosslinkers containing polyethylene glycol.
 22. The method of claim 21, wherein the first target is an antibody and the second target is a peptide respectively bound to the crosslinkers on surface-functionalized mesoporous silica nanoparticles.
 23. The method of claim 22, wherein the antibody is specific to transcriptional factors, mediators or complex in a signalling pathway and the peptide is a nucleus localization sequence (NLS)-peptide or a NLS like-peptide, so that the surface-functionalized mesoporous silica nanoparticles entering into the cell are sequestered outside of a nuclear membrane of the cell.
 24. The method of claim 22, wherein the antibody is NF-κB p65 antibody and the peptide is TAT transducing peptide.
 25. The method of claim 20, wherein the first target is superoxide dismutase and the second target is glutathione peroxidase respectively bound to the crosslinkers on surface-functionalized mesoporous silica nanoparticles.
 26. The method of claim 25, wherein the first target is TAT transducing peptide bound superoxide dismutase and the second target is TAT transducing peptide bound glutathione peroxidase respectively bound to the crosslinkers on surface-functionalized mesoporous silica nanoparticles.
 27. The method of claim 25, wherein at least one of superoxide dismutase and glutathione peroxidase is bound to the mesoporous silica nanoparticles in a denatured farm or a partially active form.
 28. The method of claim 20, wherein the surface-functionalized mesoporous silica nanoparticles are labelled with a tracking agent or a dye.
 29. The method of claim 28, wherein the dye is fluorescein isothiocyanate.
 30. The method of claim 19, wherein the first and second targets are different enzymes or catalytic mimetics capable of catalysing a cascade reaction.
 31. The method of claim 30, wherein the enzymes or catalytic mimetics are capable of catalysing the cascade reaction involved in scavenges free radicles in the cell, so as to protect the cell against ROS induced stress.
 32. The method of claim 19, wherein at least one of the first and second targets is bound to the mesoporous silica nanoparticles in a denatured form or a partially active form.
 33. The method of claim 19, wherein the first and second targets are co-encapsulated within the hollow silica nanospheres.
 34. The method of claim 33, wherein the first and second targets are different enzymes or catalytic mimetics capable of catalysing a cascade reaction.
 35. The method of claim 34, wherein the enzymes or catalytic mimetics are capable of catalysing the cascade reaction involved in scavenges free radicles in the cell, so as to protect the cell against ROS induced stress.
 36. The method of claim 34, the first target is polyethyleneimine-grafted superoxide dismutase and the second target is polyethyleneimine-grafted catalase. 